: Borrelia burgdorferi is an in vitro culturable bacterium that is the cause of Lyme disease. Its small genome contain approxnnately 1,000 chromosomal genes and 400 plasmid genes. Despite knowledge of the complete DNA sequence of the B. burgdorferi genome, identification and characterization of unique in vivo expressed B. burgdorferi virulence determinants has been delayed by the lack of expeditious and efficacious genetic systems in Borrelia. We have developed a genetic system in B. burgdorfed that consists of the extrachromosomal cloning vector, pGKI2, enhanced green fluorescent protein as a potential reporter gene, and resistance to erythromycin, kanamycin, and other antibiotics as selective markers. We have also been able to show that the bmp gene cluster is highly conserved among B. burgdorferi sensu lato strains, and that the genes of this cluster undergo environmentally modulated differential expression suggesting a potential role in virulence for these genes. The experimental protocol we propose is based on our preliminary work and framed by two hypotheses: 1) efficient molecular genetic systems can be developed for B. burgdorferi, and 2) the role of the bmp gene cluster in B.burgdorferi biology and virulence can be ascertained using these systems. With the long-term aim of identifying B.burgdorferi virulence determinants and improving our understanding of their in vivo expression and regulation, we propose the following Specific Aims: 1) Continue development and improvement of an extrachromosomal cloning system for B. burgdorferi, 2) isolate and complement B. burgdorferi bmpD, bmpC and bmpA null mutants to determine the possible role of these genes in B. burgdorferi virulence in in vitro and in vivo model systems of infection; and 3) characterize promoters and regulatory DNA sequences of bmpC using transcriptional fusions with enhanced green fluorescent protein (EGFP) and fluorescence-activated cell sorting (FACS). We expect these experiments will permit the extension of the molecular Koch's postulates to the characterization of unique aud specific molecular virulence determinants of B. burgdorferi.